Process for producing viral rna polymerase

ABSTRACT

This invention relates to a method for producing virus RNA polymerases of RNA viruses, more specifically, virus RNA polymerases of RNA viruses free of virus genomic RNA.  
     The methods described in this invention includes the procedures for preparation of cDNAs for the genes for the component proteins of RNA polymerase of an RNA virus, incorporation of the cDNA into baculovirus genome to construct recombinant virus, and the infection of insect cells with the recombinant virus to express RNA polymerase. In this method, it is recommended that all species of the recombinant viruses, each of which is designed for expressing each of the above-mentioned component protein genes of RNA polymerase, are coinfected into insect cells. Thus, cDNA is prepared for each of the component proteins of RNA polymerase and incorporated into baculovirus genome to construct recombinant virus for independently expressing the corresponding protein. In addition, the RNA viruses described above include influenza virus especially. In this method, RNA polymerase may be tagged to facilitate the subsequent purification. Furthermore, the tagged RNA polymerase described above may be purified using an adsorbent to trap the RNA polymerase at the tag. Another subject matter of this invention is to artificially prepare a complex of the component proteins of RNA polymerase of an RNA virus, or, in other words, to prepare RNA polymerase free from RNA virus genomic RNA. The RNA viruses described here include influenza virus, and the component proteins of RNA polymerase include PA, PB1 and PB2.

TECHNICAL FIELD

[0001] This invention relates to a method for producing virus RNApolymerases of RNA viruses, more specifically, a method for producingvirus RNA polymerases of RNA viruses isolated from virus genomes.

[0002] Prior art

[0003] Conventional technology related to influenza virus RNA polymerasehas focused on the development of methods to purify the polymerase fromvirus particles. Purification of the polymerase is important becauseinfluenza virus RNA polymerase can be regarded as an ultimate target forantiviral agents. From a more basic scientific viewpoint, influenzavirus RNA polymerase is an interesting material in terms of the originof its biologic evolution because, unlike DNA-dependent RNA polymerase,the RNA-dependent polymerase is considered to be involved in theproliferation of RNA genome. Despite considerable efforts, no method forefficient purification of the RNA polymerase tightly bound to viralgenomic RNA has been established to date.

[0004] Previously, we collected a complex of RNA polymerase which wasbound to genomic RNA by the centrifugation of virus particles in a highconcentration of cesium chloride solution. Moreover, we successfullydissociated the RNA polymerase-RNA complex and released both RNApolymerase and RNA in a cesium trifluoroacetate solution used bycentrifugation. The RNA polymerase, however, was found to besignificantly inactivated during the purification process. Thus, thismethod was found to be inapplicable in large-scale purifications of theRNA polymerase.

[0005] In the subsequent study, three kinds of P proteins were purifiedand mixed together in a test tube to reconstitute the RNA polymerase. Ina similar study, Summers and his colleagues of a U.S research grouputilized SDS polyacrylamide gel electrophoresis (PAGE) to separate andpurify these proteins. They have reported that a reconstituted system ofthe purified proteins showed an RNA synthesizing activity although theactivity was only limited. In our study, we purified the three kinds ofP proteins separately from cells infected with baculovirus and thenreconstituted RNA polymerase with these proteins in vitro. Similar tothe study conducted by Summers and his colleagues, we were able toconfirm that the reconstituted system showed only a limited activity.Thus, this method also appeared to be inapplicable for large-scaleproduction of RNA polymerase, and no other promising methods for thispurpose are available to date. (Honda, A., Mukaigawa, J., Yokoiyama, A.,Kato, A., Ueda, S., Nagata, K., Krystal, M., Nayak, D., and Ishihama, APurification and molecular structure of RNA polymerase from influenzavirus A/PR8. J. Biochem. 107, 624-628(1990) and Kobayashi, M., Tuchiya,K., Nagata, K., and Ishihama, A.: Reconstitution of influenza virus RNApolymerase from three subunits expressed using recombinant baculovirussystem. Virus Res. 22, 235-245(1992)).

[0006] On the other hand, Publication of unexamined Japanese PatentApplication (Kokai) No.9-121867 and Publication of unexaminedapplication (Kokai) No.11-199489 describe oligoribonucleotides asantiviral agents inhibiting protein expression induced by influenzavirus. However, these oligoribonucleotides have been unsuccessful inproving their antiviral efficacy because of the difficulty inpurification of the RNA polymerase.

[0007] Problems to be Resolved by the Invention

[0008] Conventional methods are available for isolating RNA polymerasedirectly from viruses. The content of the polymerase in a virus particleis so low that it is difficult to purify the polymerase and to removeundesirable contaminants by any of the conventional methods. Moreover,RNA polymerase in a small amounts tends to undergo rapid inactivation.Therefore, no practical methods are currently available for purificationand isolation of functional RNA polymerase.

[0009] Means to Solve the Problems

[0010] Viruses are parasitic to a host which then provides most of thesubstances required for the virus growth. Viruses, themselves, produceonly protein components of the virus genome and the virus particleaccording to their own gene information. Genomes exist as DNA in allliving organisms, but RNAs in many viruses. Therefore, a virus cannotuse the replication system of the host to replicate the genome and needsits own RNA replication system (RNA polymerase). One criticalrequirement for an antiviral agents is not to have any ill effects onthe survival and growth of the host. For this reason, together with thefact that RNA polymerase is the sole enzyme synthesized by RNA viruses,RNA polymerase has been considered as the target for which antiviralagents should be developed. The strategy of aiming development of anantiviral agent targeting the RNA polymerase has so far beenunsuccessful because, to date no effective methods have been establishedfor isolation or production of a virus RNA polymerase preparation whichis the prerequisite for such development. Our invention provides amethod for dealing with such problems and introduces a new stage ofdevelopment of antiviral agents.

[0011] The main subject of this invention is to provide a new method forproducing a virus RNA polymerase comprising the steps of preparing cDNAsfor the genes for the component proteins of an RNA polymerase of RNAvirus, integrating said cDNAs into a baculovirus genome to constructrecombinant viruses, infecting insect cells with said recombinantviruses, and expressing said cell to obtain RNA polymerase. Using thismethod, it is preferred that cDNAs each for the genes for the componentproteins of an RNA polymerase of a virus are prepared, and said eachcDNAs are integrated into each baculovirus genome respectively toconstruct recombinant viruses, and one insect cell is infected with allkinds of recombinant viruses. The RNA virus is preferably influenzavirus. In this method, RNA polymerase may be tag-labeled to facilitatethe subsequent purification. Furthermore, the RNA polymerase may bepurified by using an adsorbent to trap the tagged RNA polymerase.

[0012] Another subject of this invention is an complex, which isartificially produced, consisting of the component proteins of an RNApolymerase of RNA virus, existing isolated from RNA virus genomic RNA.The RNA virus is preferably influenza virus and the component proteinsof RNA polymerase may be PA, PB1 and PB2.

BRIEF DESCRIPTIONS ON THE DRAWINGS

[0013]FIG. 1 shows the P proteins of influenza virus expressed in insectcells infected with recombinant baculovirus.

[0014] [FIG. 1A] Sf9 cells were infected with recombinant baculovirusRBVH-PA (baculovirus having the gene for His-tagged PA protein (H-PA))at a moi (multiplicity of infection) of 2. After a 4-day culture, thecells were harvested and processed to prepare a cell lysate. The celllysate was centrifuged at 2,000 rpm for 5 min. The resulting supernatant(SUP or cytoplasm fraction) and the precipitate (PPT or nucleusfraction) were fractionated by SDS PAGE. The gel was subjected toimmuno-blotting (Western blot) using anti-PA antibodies.

[0015] [FIGS. 1B and C] Sf9 cells were coinfected with three species ofrecombinant baculoviruses (RBVPB1, RBVPB2, and RBVH-PA) at a moi(multiplicity of infection) of 2 for each virus. After a 4-day culture,the cells were harvested and processed as described above. Bothcytoplasm and nucleus fractions were immuno-blotted with anti-PB1[B] oranti-PB2[C] antibodies.

[0016]FIG. 2 shows the purification of the 3P complex from insect cellsinfected with recombination baculovirus.

[0017] [FIG. 2A] Tn5 cells were infected with recombination baculovirusRBVH-PA (Lane 2) or with all three species of recombinant baculovirus(RBVPB1, RBVPB2, and RBVH-PA) at moi of 2 for each virus, and the cellswere cultured for 4 days. The supernatant fractions (SUP) of whole celllysate were mixed with metal affinity resin, and the imidazole eluateswere fractionated by SDS-8% PAGE. The gels were stained with Coomassiebrilliant blue (CBB). Lane 1, RNP isolated from influenza virus A/PR8;lane 2, the imidazole eluate fraction from RBVH-PA-infected cells; lane3, the 3P complex isolated from cells coinfected with three species ofrecombinant baculovirus.

[0018] [FIG. 2B] Sf9 cells (lane 1) or Sn5 cells (lane 2) werecoinfected with all three species of recombinant baculovirus. After a4-day culture, the 3P complex was isolated and analyzed as describedabove.

[0019]FIG. 3 shows model template-dependent RNA synthesis activity ofthe 3P complex.

[0020] [FIG. 3A] The 3P complex purified from the recombinantvirus-infected cells or the corresponding fraction from mock-infectedcells was examined in vitro for RNA synthesis activity in incubation inthe presence of a template of v53 (lanes 1, 3, 5, and 6) or c53 (lanes2, 4, 7, and 8) and a primer of ApG (lanes 1, 2, 5, and 7) or globinmRNA (lanes 3, 4, 6, and 8). The products were separated by urea-8%PAGE.

[0021] [FIG. 3B] RNAs synthesized by the 3P complex in the presence ofthe template, v53 (lanes 1-6) or v53 (lanes 7-12), and the primer, ApG(lanes 1-3 and 7-9) or globin mRNA (lanes 4-6 and 10-12), were mixedwith an oligo(dT)₃₀ resin. The resin-bound RNAs were recovered andanalyzed by urea-6% PAGE. The migration position of a marker RNA of 100nucleotides (nt) long is shown on the left.

[0022]FIG. 4 shows template RNA-binding activity of the 3P complex.

[0023] The purified 3P complex was incubated with ³²P-labeled v53 (lanes1-3), c53 (lanes 4-6) or RNA with random sequence (lanes 7-9), and thenirradiated with a UV lamp for cross-linking. The RNA-crosslinkedproteins were analyzed by urea-8% PAGE, and the gel was exposed to X-rayfilm.

[0024]FIG. 5 shows capped RNA-binding and cleavage activities of the 3Pcomplex.

[0025] [FIG. 5A] The purified 3P complex (12 pmol/ml or 3 μg/ml; lane 2,5 μl; lane 3, 10 μl) and the isolated RNP (lane 4) were assayed for thecapped RNA-binding activity. RNA with radioactivity of ³²P only at thecapped position was mixed with the 3P complex or RNP and then irradiatedwith a UV lamp. After digestion with ribonuclease T1 and ribonuclease A,the RNA-cross-linked proteins were analyzed by urea-8% PAGE. The gel wasexposed to X-ray film.

[0026] [FIG. 5B] RNP (lane 1), the 3P complex (lanes 2-4), and thecorresponding fraction from mock-infected cells (lane 5) were assayedfor the capped RNA endonucease. Each fraction was mixed with cappedpoly(A) labeled with ³²P only at cap-1 in the absence of (lanes 1-2) orpresence of either 1 pmole of v53 (lanes 3 and 4) or 1 pmole of c53(lane 5) template at 30° C. for 30 min. The resulting incubation mixturewas analyzed for RNA. The gel was exposed to an imaging plate overnight,and the plate was analyzed with a BAS2000 image analyzer (Fuji).

DESCRIPTION OF THE EMBODIMENTS

[0027] RNA viruses can be classified into two categories. RNA viruses ofone category can synthesize RNA polymerase by encoding the genomic RNAand infecting the host. In other words, RNA viruses of this category canutilize the genome as the template (mRNA) in the protein synthesis, andthus are called “plus-strand RNA viruses”. RNA viruses of the othercategory can not utilize the genome as the template in the proteinsynthesis, but transcribe the genomic information onto a complementarychain to use the chain as the mRNA. Thus, RNA viruses of this lattercategory are called “minus-strand RNA viruses”. Host living organisms donot have any enzyme to catalyze transcription of the genomic RNA tocomplementary RNA. RNA viruses of this latter category, however, haveRNA polymerase in the virus particle. RNA viruses of this lattercategory include those of Paramyxoviridae, Orthomyxoviridae,Rhabdoviridae, Bunyavirus, and Arenaviridae. This invention can beapplied to production of RNA polymerase of all viruses of this category.

[0028] When this invention is applied to the production of RNApolymerase of the influenza virus, the procedures are as follows:

[0029] (1) Preparation of three species of recombinant baculoviruses,each of which independently expresses one of the subunits, PB1, PB2, andPA, of influenza virus RNA polymerase. cDNA for PB1 or PB subunit isincorporated into baculovirus genomic DNA by homologous recombination asfollows. First, these cDNAs are incorporated into the intermediatevector of E. coli to confirm their expression in E. coli cells. Theseintermediate vector DNAs are introduced with the baculovirus DNA intohost cells of an established insect cell strain by electroporation orthe lipofectin method. Homologous recombination is induced in the cells,and the resulting transformed baculoviruses containing the genes for PB1and PB2 are isolated. On the other hand, cDNA of the other subunit, PA,is introduced into a downstream region from a histidine (His) tag label(HAT) in the pHAT vector containing the HAT label to express the PAprotein in a fused form with a sequence rich inHis residues at theN-terminal and thus to facilitate the purification of the RNA polymerasefrom the cells expressing the polymerase. This intermediate vector isinserted into baculovirus DNA by homologous recombination in a mannersimilar to that described above for insertion of PB1 and PB2.

[0030] (2) Reconstitution of influenza virus RNA polymerase with itsthree P protein subunits simultaneously expressed by recombinantbaculoviruses-infected insect cell system

[0031] Insect cells are infected with three species of recombinantbaculoviruses, each of which is constructed for expression of one of thethree P protein subunits of influenza virus RNA polymerase. Theexpression of each subunit is confirmed by Western blot with thecorresponding antibody (an immunoassay). The cells are homogenized witha homogenizer to prepare a nucleus fraction. Nuclei are treated with adetergent and centrifuged at 50,000 rpm for 2 hr. The resultingsupernatant is mixed with TALON metal affinity resin (an adsorbent) totrap proteins labeled with a His tag. The resin adsorbing proteins aretreated with 100 mM imidazole to reduce the interaction between theresin and the His tag and thus to elute the proteins. The eluate isfractionated by SDS PAGE to analyze the expressed P protein subunits byWestern blotting. In this invention, PA alone is labeled with the Histag. Nevertheless, the eluate contains not only His tagged PA, but alsoPB1 and PB2. This indicates that the three P protein subunits areobtained as a complex form by the method described in this invention.

[0032] (3) Purification of the influenza virus RNA polymerase expressedin the insect cell system and measurement of its activity

[0033] A crude extract is obtained from a homogenate of recombinantbaculovirus-infected insect cells. From the extract, reconstituted RNApolymerase is purified as described above. The purified specimen isfractionated by SDS PAGE. Proteins in the gel are stained with Coomassieblue to confirm the purity of the P proteins. The method described inthis invention has confirmed to offer a purified preparation offunctional P protein complex in terms of the following four activities:

[0034] (1) template RNA-dependent RNA synthesis activity

[0035] (2) primer-dependent RNA synthesis activity

[0036] (3) host-derived mRNA cap (cap-1) structure-recognizing andbinding activity

[0037] (4) activity to cleave cap (cap1) structure-containing RNA at aspecific site

[0038] RNA polymerases of viruses in the categories described above inthis invention can be produced by the method described above.

[0039] Pratical examples are shown below to demonstrate this invention,but not to restrict this invention to the examples.

EXAMPLES

[0040] The practical examples were carried out by using the followingmaterials.

[0041] (i) Bac-to-Bac vector (GIBCO-BRL Inc.) for construction ofrecombination baculoviruses

[0042] (ii) Insect cells of established cell line (including Sf9 andTn5) for expression of virus proteins

[0043] (iii) TALON metal affinity resin (CLONTECH Inc.) for purificationof the proteins expressed by insect cells

[0044] (iv) Reagents for measurement of RNA polymerase activity

[0045] The substrates for RNA synthesis (ATP, GTP, CTP, and UTP)(Pharmacia Inc.), and ApG and globin mRNA to be used as the primer

[0046] (v) Reagents for preparing template RNA

[0047] Vector pV53 for preparing template RNA and T7 RNA polymerase(TAKARA)

[0048] (vi) Reagents for measurement of capped RNA cleavage activity

[0049] Labeling agent for the RNA cap-site, [α-³²P]GTP, and cappingenzyme

[0050] Test Procedures

[0051] The insect cell lines established from Spodoptera frugiperda(Sf9) and Trichoplusia ni (Tn5) were used for infection with recombinantbaculoviruses. Autographa california nuclear polyhedrosis virus (AcNPV)was used for construction of recombinant viruses.

[0052] Recombinant baculoviruses for expression of PB1 and PB2 wereconstructed as described in the previously published report (KogayashiM., Tsuchiya K., Nagata K., and Ishihama A. (1992) Virus Res. 22235-245). For the construction of recombinant virus for expression ofPA, cDNA for PA was amplified by polymerase chain reaction (PCR) andinserted between the NcoI and BglII sites of pAcHLT-B (Phamigen). Theresulting plasmid DNA (pAcHLTPA) was linearized and co-transfected withbaculovirus DNA into Sf9 insect cells by the liposome method. After72-hr culture at 27° C., the supernatant was harvested and therecombinant viruses contained in the supernatant were infected into Sf9cells. The culture supernatant was harvested, and the titer ofrecombinant virus (RBVH-PA) was determined by the plaque assay. Thetiter of the virus preparation was 10⁸ PFU/ml.

[0053] Tn5 or Sf9 cells were coinfected with the three species ofrecombinant baculovirus at a moi of 2 for each. After a4-day culture forthe co-infection, 10⁸ cells were collected from the culture andsuspended in 5 ml of the cell disruption buffer, 10 mM HEPES buffer (pH7.6) containing 10 mM KCl, 1.5 mM MgCl₂, 2 mM dithiothreitol (DTT), 0.1%TritonX-100 and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma). Thecells suspension was homogenized by using a Dounce homogenizer andcentrifuged at 2,000 rpm for 5 min to recover nuclei. The nuclei werehomogenized in 3 ml of the nucleus extraction buffer, 10 mM sodiumphosphate buffer (pH 7.0) containing 500 mM NaCl and 20% glycerol. Thehomogenate was incubated with stirring in an ice bath for 30 min andthen centrifuged at 40,000 rpm for 2 hr. The resulting supernatant(nucleus extract) was mixed with metal affinity resin (Clontech) andincubated for 1 hr at 4° C. with constant rotation. The resin was washedwith the washing buffer, 50 mM sodium phosphate buffer (pH 7.0)containing 300 mM NaCl and 5 mM imidazole until UV monitoring of theeluate showed no detectable proteins in the eluate. The resin was theneluted with the elution buffer containing 100 mM imidazole. The elutedproteins were analyzed by SDS-8% PAGE, and the gels were stained withCoomassie brilliant blue (CBB).

[0054] The P proteins separated by SDS-PAGE were electro-blotted ontoPVDF membranes in 10 mM CAPS buffer (pH 11) containing 10% methanol. Theblotted filters were incubated with anti-PA, anti-PB1, and anti-PB2antibodies for 1 hr at 37° C. and then with peroxidase-conjugatedanti-rabbit IgG. The peroxidase activity was visualized by the reactionwith 3,3′-diaminobenzidine tetrahydrochloride (DAB) (DOJIN) to detectthe P proteins. The anti-P protein antibodies used in the detection ofthe expressed P proteins had been raised in rabbits against the purifiedP proteins over expressed in the culture of E. coli cells.

[0055] RNA synthesis was carried out for 60 min at 30° C. in 50 μl of astandard reaction mixture in 50 mM HEPES/KOH buffer (pH 7.6) containing100 mM NaCl, 5 mM magnesium acetate, 2 mM DTT, 0.25 mM each of ATP, GTP,and CTP, 4 μM UTP, 10 μCi [α-³²P]UTP, 0.25 mM ApG or 250 ng globin mRNA,1 unit RNasin (Promega), and 1 pmol of v53 or c53 model RNA template.The transcripts were analyzed by 10% PAGE in the presence of 7M urea.The gels were exposed to imaging plates and then analyzed with a BAS2000image analyzer (Fuji). The model templates (v53 and c53) weresynthesized by transcribing pV53 or pC53 plasmid DNA using T7 RNApolymerase.

[0056] Radioactive vRNA and cRNA were synthesized by transcribing pV53and pC53 DNA, respectively, by using T7 RNA polymerase in the presenceof radioactive substrates. In an RNA-binding reaction mixture of 50 mMHEPES/KOH buffer (pH 7.8) containing 100 mM NaCl, 5 mM magnesiumacetate, 2 mM DTT, and 10 μg tRNA, 10 μl of the 3P complex (a complexconsists of 3 species of protein) and 10,000 cpm of radioactive v53, c53or random RNA (TMV 3′-terminal sequence) were incubated in a finalvolume of 50 μl for 30 min at 30° C. The reaction mixture was irradiatedwith a UV lamp for 30 min. After the addition of anti-PB1, the mixturewas further incubated at 37° C. for 1 hr. The mixture was incubated withprotein A-Sepharose (Pharmacia) for 2 hr in an ice bath to recover theresulting antigen-antibody immunocomplexes as protein A-Sepharosecomplexes. The protein A-Sepharose complexes were washed once with PBSand then subjected to SDS-8% PAGE. The gels were analyzed as describedabove.

[0057] Cross-linking of capped RNA to the P proteins was carried out asdescribed in the previously published report (Honda A., Mizuno K., andIshihama A. (1998) Virus Res. 55 199-206). In brief, globin mRNA wasdecapped and recapped for one cycle by treating with vaccinia virusguanylyltransferase (Gibco BRL) in the presence of [α-³²P]GTP. Therecapped globin mRNA with a cap structure labeled with ³²P (about 1,000cpm) only at the 5′-position was incubated with the 3P complex or viralnucleoprotein (RNP) in 50 mM HEPES/KOH buffer (pH 7.6) containing 100 mMNaCl, 5 mM magnesium acetate, 2 mM DTT and 1 unit of RNasin (Promega) ina final volume of 50 μl at 30° C. for 30 min. The mixture was irradiatedwith a UV lamp for 30 min. The RNA-cross-linked proteins were digestedwith RNase A and RNase T1 and then incubated with anti-PB2 at 37° C. for1 hr. The reaction mixture was incubated with protein A-Sepharose(Pharmacia) for 2 hr in an ice bath to precipitate the antigen-antibodyimmunocomplexes formed. The precipitate was washed once with phosphatebuffered saline (PBS) and then fractionated on an SDS-8% gel byelectrophoresis. The gel was analyzed as described above.

[0058] The purified 3P complex was incubated in 50 mM HEPES/KOH buffer(pH 7.8) containing 100 mM NaCl, 2 mM DTT, 0.3% Triton X-100, 0.25 mg/mlBSA, and capped globin mRNA or poly(A) (with a cap structure labeledwith about 2,000 cpm of ³²P only at the 5′-position) for 30 min at 30°C. in a final volume of 50 μl. The reaction products were extracted withphenol-chloroform, precipitated with ethanol, and analyzed by 12% PAGEin the presence of 7 M urea. The gel was analyzed as described above.

[0059] Oligotex (dT)₃₀ (Takara) was added to the reaction mixture. Thereaction mixture was heated at 75° C. for 10 min. The heat-treatedmixture was placed in an ice bath for 5 min, and 5 M NaCl was then addedto the mixture to give a final concentration of 500 mM. The mixture wasincubated at 37° C. for 10 min and then centrifuged at 2,000 rpm for 5min. The precipitate was washed with 10 mM Tris-HCl buffer (pH 7.5)containing 1 mM EDTA, 0.5 M NaCl, and 0.05% SDS and resuspended in 0.1ml of sterilized distilled water. RNA was extracted withphenol-chloroform, precipitated with ethanol, and then analyzed by PAGEin the presence of 7 M urea.

[0060] Test Results

[0061] For the expression of the three different P protein subunits ofinfluenza virus RNA polymerase, recombinant baculoviruses wereconstructed using cDNAs for vRNA segments 1, 2, and 3, containing thegenes for PB1, PA, and PB2 proteins, respectively. cDNA for PA wasinserted into the pAcHLT vector to express the PA protein as a fusedform with a histidine (His) tag sequence of 40 amino acids in length atits N-terminal. This procedure facilitated the purification of theresulting 3P complex. From the fact that the N-terminal sequence of PAwas not involved in subunit-subunit contact, the addition of the His tagwas presumed to have no inhibitory effects on the assembly and activityof RNA polymerase. The construction of recombinant baculoviruses forproduction of PB1 and PB2 was described in the previously publishedreport (Kogayashi M., Tsuchiya K., Nagata K., and Ishihama A. (1992)Virus Res. 22 235-245).

[0062] To confirm the expression of the P proteins, recombinantbaculoviruses were infected into Sf9 cells under various conditions, andthe whole cell lysates were analyzed by immunostaining with specificpolyclonal antibodies against each P protein. In the case of Sf9 cellsinfected with the recombinant baculovirus RBVH-PA alone, a band showinga cross reaction with anti-PA was detected in both cytoplasm and nucleusfractions by SDS-8% PAGE (FIG. 1A, lanes 2 and 3). The migration of H-PA(His-tagged PA) was slightly slower than that of authentic PA withoutHis-tag (FIG. 1A, lane 1). The immunostaining detected no bands in theextracts of mock-infected cells (FIG. 1A, lanes 4 and 5). Sf9 cellsinfected with the recombinant baculovirus RBVH-P alone expressed neitherPB 1 nor PB2. In the extract of Sf9 cells coinfected with all the threerecombinant viruses, RBVPB1, RBVPB2, and RBVH-PA, the expression of PB1and PB2 in addition to H-PA was demonstrated by immunostaining withanti-PB1 and anti-PB2 (FIGS. 1B and C). PB1 and PB2 were again recoveredin both of the cytoplasm and nucleus fractions.

[0063] In order to examine whether the three viral P proteins formcomplexes in insect cells, Tn5 cells were coinfected with the threespecies of recombinant baculoviruses (at moi 2 for each virus). Afterthe cell lysate was examined by SDS-8% PAGE, all three P proteins weredetected by immunostaining with the corresponding antibodies. Themajority of each was recovered mostly in the nucleus fraction (FIG. 1).To purify P protein complex, the nucleus extract was centrifuged at40,000 rpm for 1 hr and the supernatant was directly subjected to metalaffinity resin purification. The material eluted with 100 mM imidazolewas fractionated by SDS-8% PAGE and the gel was stained with Coomassiebrilliant blue (CBB). Three bands were detected (FIG. 2A, lane 3). Thesebands showed cross-reactivity with anti-PB1, anti-PA and anti-PB2antibodies in the order of migration. SDS-PAGE showed that both PB1 andPB2 co-migrated with the corresponding P protein associated with viralRNP cores (FIG. 2A, lane 1). On the gel, the H-PA migrated as fast asthe recombinant H-PA recovered from RBVH-PA virus-infected cells (FIG.2A, lane 2), but more slowly than the RNP-associated untagged PA (FIG.2A, lane 1). Thus we conclude that at least some of the PB1 and PB2molecules are associated with His-tagged PA protein.

[0064] In order to construct a high-level expression system for theseinfluenza virus P proteins we compared the levels of these proteinsexpressed in cells of two insect cell lines, Tn5 and Sf9, which werecultured under the same conditions at 27° C. After the purification byusing a metal affinity resin, all three P proteins were detectable evenby CBB staining of SDS-gel (FIG. 2B). From the staining intensity, itindicates that the 3P complex thus isolated contained the three Pproteins in equal amounts. Since the level of the 3P complex expressedin the Tn5 culture was higher than in the Sf9 culture, Tn5 was used forpurification of the 3P complex in a large scale. From the CBB stainingintensity, the yield of the 3P complex was estimated to be about 5 μgfrom 1×10⁸ virus-infected Tn5 cells.

[0065] The influenza virus RNA polymerase solubilized from viral RNPcores shows RNA synthesis activity if exogenously added model RNAtemplates which carry terminal conserved sequences of viral RNAsegments. The RNA synthesis activity of the purified 3P complex wasexamined using two model templates (v-sense (minus-strand) v53 andc-sense (plus-strand) c53) and two alternative primers (globin mRNA anddinucleotide ApG). When v53 was used as a template, RNA products weredetected in both ApG- and mRNA-primed reaction (FIG. 3A, lanes 1 and 3).One of the transcripts from the mRNA-primed reaction (lane 3) was longerthan the ApG-primed transcript by about 12 nt (lane 1). The nucleusextract of mock-infected cells was unable to synthesize RNA from eithertemplate (FIG. 3A, lanes 5-8). These results indicate that the detectedRNA synthesis is mediated by the 3P complex formed in insect cellsinfected with recombinant virus, but not by any cellular enzymes in theinsect cells. Scince the 3P complex is unable to synthesize any RNA whengiven RNA template with random sequences, this indicates that the 3Pcomplex, similar to the native viral RNA polymerase, recognizes vRNA ina specific manner.

[0066] When c53 was used as template, RNA products were detectedparticularly in the presence of ApG primer (FIG. 3A, lane 2). The sizeof the major transcript from this cRNA-directed and ApG-primed reactionwas, however, shorter than the 53 nt template. This was probably due tothe internal initiation at an as yet unidentified ApG-binding sitewithin the c53 RNA. The activity of globin mRNA-primed transcription waslow, and no clear bands of transcripts were detected at least under theconditions employed (FIG. 3A, lane 4). These results indicate that onlyv-sense RNA may be able to direct capped RNA-primed transcription.

[0067] Influenza virus RNA polymerase is interconvertible betweentranscriptase and replicase, but the capped RNA-primed initiation of RNAsynthesis is a unique characteristic of the transcriptase. On the otherhands, the replicase form of RNA polymerase is considered to be involvedin the initiation of de novo RNA synthesis without using primers becausevRNA in virus particles retains 5′-triphosphate. The 3P complex purifiedfrom insect cells infected with recombinant baculovirus did not catalyzeRNA synthesis in the absence of primers. Therefore, we propose that the3P complex represents the transcriptase form of viral RNA polymerase.

[0068] Influenza virus RNA polymerase recognizes specific sequenceslocated at 5′- and 3′-terminals conserved sequences of vRNA and cRNA,which act as transcription promoter and replication origin, and itsbounding to these sequences exerts its intrinsic activity of enzyme.Accordingly, the binding of the 3P complex to vRNA and cRNA wasexamined. The 3P complex was incubated with a radio-labeled modeltemplates, v53 and c53, and random RNA of a similar size at 30° C. for30 min in the transcription assay mixture without substrates, thenirradiated with a UV lamp for 30 min to promote cross-linking.Immediately after the UV irradiation, the mixture was subjected to RNasedigestion. The digested products were immunoprecipitated with acombination of anti-PB 1 and protein A. The immuno-precipitates wereanalyzed by SDS-8% PAGE. Both vRNA (FIG. 4, lanes 2 and 3) and cRNAmodel templates were cross-linked to the 3P complex (FIG. 4, lanes 5 and6), but random RNA (FIG. 4, lanes 8 and 9) was not. These resultsindicate that the 3P complex formed in insect cells, similar to thenatural virus RNA polymerase, is able to specifically recognize bothvRNA and cRNA.

[0069] One unique feature of influenza virus growth is that host cellcapped RNA is used as a source of primers for initiation of viraltranscription. Even more remarkably, the influenza virus RNA polymeraseitself is able to cleave capped RNA at specific positions near the5′-cap structure. Therefore, the finding that globin mRNA could serve asa primer for transcription by the 3P complex indicates that the 3Pcomplex has both capped RNA binding and cleavage activities. To confirmthis prediction, we examined next by the binding activity of 3P complexto capped RNA. For this purpose, rabbit globin mRNA recaped with[α-³²P]GTP was prepared using vaccinia virus guanylyltransferase. The 3Pcomplex or viral RNP was then incubated with recapped RNA (with ³²P onlyat the cap-1 structure). Immediately after the incubation, the mixturewas irradiated with a UV lamp for cross-linking. After subsequentdigestion with RNase A and RNase T1, the 3P complex wasimmunoprecipitated with a combination of anti-PB2 serum and protein A,and the immuno-precipitates were analyzed by SDS-8% PAGE. The nucleusextract from mock-infected cells produced no radioactive bands at the Pprotein positions (FIG. 5A, lane 1). Both of the 3P complex (FIG. 5A,lanes 2 and 3) and RNP (FIG. 5A, lane 4), however, revealed ³²Pradioactivity at the position corresponding to the PB2 band. Theseresults suggest that the 3P complex possesses the capped RNA-bindingactivity. In the products extracted from insect cells infected withrecombinant baculovirus, radioactivity was also detected in at least oneadditional band migrating faster than PB2. This band, however, was alsodetected in the nucleus extract from mock-infected cells, indicatingthat it corresponds to a fast migrating host (insect cells) proteinhaving a binding activity to the capped RNA.

[0070] Next, we examined for its capped RNA cleavage activity, using thepurified 3P complex. In this experiment, both globin mRNA and cappedpoly(A), both with ³²P only at the cap-1 structure, were used as thesubstrates. Capped poly (A) is a good substrate for endonucleolyticcleavage by RNP. As expected, incubation with RNP resulted in cleavageof the capped poly(A) into fragments of 10-13 nts in length (FIG. 5B,lane 1). The capped RNA was also cleaved by the purified 3P complex inthe presence of v53 template (FIG. 5B, lane 4), but not in the absenceof template RNA (FIG. 5B, lane 3). No cleavage of the capped RNA wasalso observed with the corresponding extract from mock-infected cells(FIG. 5B, lane 2). It has been previously observed that capped RNAendonuclease associated with RNA polymerase is activated via binding tothe template RNA (Hagen, M., Chung, T. D., Butcher, J. A., and Krystal,M. (1994) J. Virol. 68, 1509-1515; Li, M. L., Ramirez, B. C., and Krug,R. M. (1998) EMBO J. 17, 5844-5852). To our surprise, however, theenhancement of capped RNA endonuclease was observed only with v53template. c53 was virtually inactive in the activity this regulatoryfunction of RNA polymerase activation (FIG. 5B, lane 5). These resultsdemonstrate for the first time that the 3P complex discriminates betweenv-sense and c-sense RNAs.

[0071] If the 3P complex formed in insect cells possesses the catalyticactivity as a transcriptase, these may include an activity ofpolyadenylation coupled to vRNA transcription. To test this possibility,globin mRNA-primed vRNA-transcription products were characterized indetail. Analysis of the RNA products by 10% PAGE revealed diffuseradioactive bands above the 65-nt-long transcript. To examine whetherthese slowly migrating RNAs carry poly(A) sequences added to the newlysynthesized RNA, in vitro transcripts were mixed with an oligo(dT)₃₀resin, and the resin-bound RNA was eluted by heat-treatment in thepresence of high concentrations of salt to eluted RNAs. The eluate wasanalyzed by 6% PAGE in the presence of 7 M urea. As shown in FIG. 3B, aradioactive RNA was detected, which migrated more slowly than a sizemarker of about 100 nt in length. The average size of oligo(dT)₃₀-boundRNAs was greater for mRNA-primed transcripts (FIG. 3B, lanes 5 and 6)than for ApG-primed transcripts (FIG. 3B, lanes 2 and 3). These resultsindicate that at least some of the RNA products formed on the vRNA modeltemplate in the presence of either ApG or mRNA primers contain poly(A)sequences. On the other hand, most of the cRNA-directed transcripts didnot bound to the oligo(dT)₃₀ resin. After treatment of the vRNAtranscripts with RNase A and RNase T1, some nuclease-resistant RNAmoieties were detected by gel electrophoresis. These moieties were mostlikely to be poly(A) sequences. Such polyadenylation of vRNA-directedtranscripts is another characteristic and unique function associatedwith the transcriptase.

[0072] Advantageous Effect of the Invention

[0073] Efforts to establish prevention and treatments of influenza havebeen focused mainly on the development of the immuno-therapy using theantigens against the whole particles or a component protein(s) of thevirus and the exploratory studies on agents to interfere withphysiological functions of the components of the virus. Among thetargets examined in such efforts, the only target left is RNApolymerase. The method for RNA polymerase production in a large-scaleestablished in this invention provides the base for the mass-screeningof inhibitors of RNA polymerases and drug discovery from substrateanalogues for the RNA polymerase-mediated reactions. Moreover, RNApolymerase production facilitates the protein structure identificationof RNA polymerases and even further drug discovery based on theidentified structures.

1. (revised) a method for producing an RNA polymerase of influenza virus comprising the steps of preparing cDNAs each for the genes for three component proteins (PA, PB 1 and PB2) of an rna polymerase of influenza virus, integrating said each cDNAs into each baculovirus genome respectively to construct three kinds of recombinant viruses, infecting one insect cell with said three kinds of recombinant viruses, and expressing said cell at the same time to obtain RNA polymerase.
 2. (Deleted)
 3. (Revised) The method of claim 1 or 7, wherein the subunit PA of said RNA polymerase is tag-labeled at the N-terminal.
 4. The method of claim 3, further comprising a step of purifying the RNA polymerase by using an adsorbent to trap the tagged RNA polymerase.
 5. (Revised) An RNA polymerase, which is artificially produced and is in the form of a complex consisting of three component proteins (PA, PB1 and PB2) of an RNA polymerase of influenza virus, existing isolated from RNA virus genomic RNA, and having an RNA polymerase activity of influenza virus.
 6. (Deleted)
 7. (Added) A method for producing an RNA polymerase of influenza virus comprising the steps of preparing cDNAs each for the genes for three component proteins (PA, PB1 and PB2) of an RNA polymerase of influenza virus, integrating said each cDNA into each baculovirus genome respectively to construct three kinds of recombinant viruses, infecting one insect cell with said recombinant viruses, expressing said cell at the same time, and contacting the resultant complex of the component proteins with an influenza virus or its essential moiety. 